The invention relates to the preparation of synthetic membranes, such as biocompatible membranes, and matrices.
Synthetic membranes have many uses, such as in the emerging field of tissue engineering. In general, engineered tissue analogs, often composed of cultured cells, biomaterials, or composites combining cells and biomaterials, have achieved some clinical success, for example, as substitutes for skin and cartilage.
Significant effort has been devoted to producing biocompatible scaffolds with defined pore sizes that help ensure proper cell-cell contacts, cell-matrix interactions, and to preserve cellular function. For example, collagen sponges and foams, cylindrical poly(l-lactic acid) (PLA) devices, and polyglycolic acid (PGA) fibers processed into porous, nonwoven mesh matrices, have been made to provide substitute skin, nerves, and cartilage. Although sponges, foams, and matrices are useful for the fabrication of relatively large, three-dimensional tissue analogs, their use is limited for the creation of thin membranes such as the basal lamina, a membranous layer of connective tissue found in many organs and tissues.
The basal lamina or basement membrane is a thin membranous layer of connective tissue that underlies all epithelial cell sheets and tubes. For example, a basal lamina separates the endothelial cell layer of blood vessels from the underlying tissue and a basal lamina separates the epidermis from the underlying dermal tissue. A basal lamina also surrounds individual muscle cells, fat cells, and Schwann cells of nerve fibers. The basal lamina separates these cells and cell sheets from the underlying or surrounding connective tissue. In other locations, such as in the kidney glomerulus and lung alveolus, a basal lamina lies between two cell sheets and functions as a highly selective filter. Basal laminae serve more than structural and filtering roles. They are also able to determine cell polarity, influence cell metabolism, organize the proteins in adjacent plasma membranes, induce cell differentiation and serve as specific highways for cell migration. The basal lamina can also serve as a selective barrier to the movement of cells. The lamina beneath an epithelium, for example, usually prevents fibroblasts in the underlying connective tissue from contacting the epithelial cells, but does not prevent the movement of immune cells in and out of the epidermis, nor does it prevent the innervation of the epidermis. During wound healing as well as during normal development, the basal lamina acts as a guide and template that helps control cell migration and differentiation.
The invention is based on the discovery that a membrane can be manufactured to have a highly controlled and complex three-dimensional topography by using microfabrication techniques. Similarly, a matrix can be manufactured to have a highly controlled and complex three-dimensional surface topography. The new microfabricated membranes and matrices can be prepared of man-made as well as natural materials, such as materials found in naturally occurring membranes, and thus can be made in the form of tissue substitutes or analogs, such as basal lamina analogs.
Given the important role of the basal lamina membrane in many different tissues and organs, the ability to produce basal lamina analogs with a controlled and complex three-dimensional topography has numerous applications in tissue engineering and the manufacture of artificial organs. Because of their carefully controlled and defined topographies and high surface areas, synthetic, microfabricated membranes can be used, for example, in air and water filters, cell culturing devices, and blood dialysis devices.
In general, the invention features microfabricated membranes including a sheet of conforming material that comprises a defined, three-dimensional topography, e.g., invaginations and/or projections. The membranes can be made of conforming materials such as gelatins, collagens, polyurethanes, polylactic acids, TEFLON(copyright), polystyrenes, epoxy resins, methacrylates, polycarbonates, silicones, non-collagenous proteins, or polysaccharides. The membranes can also be made of copolymers, such as blends of polylactic acid and polyglycolic acid, or natural materials such as proteoglycans and glycosaminoglycans, as well as blends of natural and synthetic materials. The membranes can be, e.g., from 1 to 500, or 1 to 5, 10, 15, 20, 35, or 50 microns thick. The topographic features can have a height or depth of, e.g., 1.0 to 1000 microns, or 10 or 20 to 100 or 200 microns. The topographic features can have a width of, e.g., 1.0 to 500 microns, or 5, 10, or 20 to 100, 200, or 300 microns. The membranes can have a controlled porosity or permeability.
The invention also features new basal lamina analogs that include a microfabricated membrane, wherein the membrane is 1 to 50 microns thick, and the three-dimensional topography is defined to mimic the three-dimensional topography of a natural basal lamina.
In another aspect, the invention also features dermal or tissue analogs that include a polymeric matrix and a basal lamina analog fixed, e.g., laminated, to a surface of the polymeric, e.g., protein such as collagen, matrix. The polymeric matrix can include type I or type IV collagen and a glycosaminoglycan (GAG). The polymeric matrix can also be non-proteinaceous, and include, e.g., hyaluronic acid. The polymeric matrix can include any of the conforming materials mentioned herein that can be used to form the membranes.
The invention also features tissue substitutes including a dermal or tissue analog, and mammalian, e.g., human, canine, feline, bovine, equine, porcine, or ovine cells, e.g., epithelial cells, grown on and/or in the dermal analog. When the epithelial cells are keratinocytes, the tissue substitute is a skin substitute. In some embodiments, the mammalian cells are engineered to include a nucleic acid construct that encodes a heterologous polypeptide, or a therapeutic protein, a growth factor, a wound healing factor, or a hormone.
The invention also features a method of preparing a microfabricated membrane comprising a defined, three-dimensional topography, by preparing a master plate comprising a defined, three-dimensional pattern; transferring the pattern or a negative of the pattern to a membrane material; and allowing the membrane material to solidify, e.g., polymerize, harden, or gel, to form the microfabricated membrane, wherein the membrane has a defined, three-dimensional topography that is substantially the same as the three-dimensional pattern of the master plate or a negative of the master plate pattern.
The pattern can be transferred from the master plate to the membrane material by applying the material directly to the master plate, to produce a microfabricated membrane that has a defined, three-dimensional topography that is substantially the same as a negative of the three-dimensional pattern of the master plate. Alternatively, the pattern can be transferred from the master plate to the membrane material by coating the master plate with a liquid or semi-solid conforming material, e.g., polydimethyl-siloxane silicone elastomer (PDMS); allowing the conforming material to solidify, and removing the conforming material from the master plate to form a negative replicate that comprises a negative of the master plate pattern; applying a membrane material to the negative replicate; and allowing the membrane material to solidify to form the microfabricated membrane and removing the membrane from the negative replicate, wherein the membrane has a defined, three-dimensional topography that is substantially the same as the three-dimensional pattern of the master plate. The invention also includes microfabricated membranes prepared by the new methods.
In yet another embodiment, the invention covers a method of preparing a microfabricated tissue analog comprising a defined, three-dimensional surface topography, by preparing a master plate comprising a defined, three-dimensional pattern; transferring the pattern or a negative of the pattern to a matrix material; and allowing the matrix material to solidify to form the microfabricated tissue analog, wherein the analog has a defined, three-dimensional surface topography that is substantially the same as the three-dimensional pattern of the master plate or a negative of the master plate pattern. The invention also includes tissue analogs made by this method.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The invention provides several advantages. For example, the invention provides virtually unlimited design possibilities for the precise control of the three-dimensional topography of thin, synthetic membranes, such as biologically active membranes. Moreover, the new microfabricated, synthetic membranes can be prepared in the form of novel skin substitutes, which have applications in the treatment of burns, plastic surgery, ulcers, and gene therapy.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.